Current automated DNA sequencing methods use fluorescence detection of labeled DNA sequencing fragments. One method is to form four sets of DNA sequencing fragments terminating in G, A, T or C where each set is labeled with the same fluorophore, and then mn the sequencing fragment sets in adjacent lanes in a slab gel electrophoresis apparatus. Various apparatus have been suggested for scanning the gel to monitor said fragments as or after they move through the gel. In copending application Ser. No. 07/531,900 filed Jun. 1, 1990, U.S. Pat. No. 5,091,652, incorporated herein by reference, there is described a laser-excited confocal fluorescence gel scanner which provides enhanced detection of fluorescently labeled DNA sequencing fragments separated on a slab gel.
The detection system uses an epi-illumination format where the laser power is focused on the sample by a microscope objective followed by confocal detection. However, lane-to-lane variations in the migration velocity of the DNA fragments make it difficult to deduce the correct alignment of the bands in the four sequencing lanes. The throughput is reduced because of the need for running four lanes to detect the four sets of DNA sequencing fragments terminating in G, A, T and C.
A solution proposed to overcome these drawbacks is to label each sequencing fragment set with a different fluorophore, and then to perform the electrophoresis operation in only one lane. This requires a multi-color detection system and dyes that do not alter the mobility of the fragments relative to one another. A method and apparatus for sequentially scanning four colors in multiple lanes in a slab gel is described in U.S. Pat. No. 4,811,218 and by Smith, et al., Nature 321,674 (1986). An alternative method using four different dye labeled dideoxy terminators along with two-color detection has been described in U.S. Pat. No. 4,833,332 and by Probet et al. Science 238, 336 (1987).
Capillary electrophoresis is emerging as a high-speed DNA sequencing method. In copending application Ser. No. 07/840,501 filed Feb. 24, 1992, U.S. Pat. No. 5,274,240, there is described an automated sequencing apparatus which employs an epi-illumination format where a laser is focused to a small volume by a microscope objective and fluorescence emitted from said volume is gathered by the same objective followed by confocal detection. An array of side-by-side parallel capillaries is sequentially and periodically moved past the focal volume or vice versa to cause and detect fluorescence in labeled DNA sequencing fragments within the capillaries. The capillary array electrophoresis scanner is described in said application for use in a one-color, single-channel detection system where each set of DNA sequencing fragments is separated in a separate capillary or in a four-color, four-channel detection system where each set of fragments is labeled with a different fluorophore for separation and detection in only one capillary.
It is very difficult, in practice, to find four dyes of exactly the same electrophoretic shift. Therefore, it becomes necessary to perform complicated shift corrections before the sequence can be read. Four-color detection has been described in connection with capillary electrophoresis by Smith and coworkers using a simultaneous four-color detection system where the signal is split between each of four channels (Nucleic Acids Research 18, 4417-4421 (1990)). This is satisfactory, but the signal-to-noise ratio is reduced because the signal is split between four different channels, and the problem of maintaining equal band shifts for each of the sets of labeled sequencing fragments using different dyes must still be resolved.